The objective of this grant proposal is to understand the process of amelogenesis. To achieve our goals, we propose initially to characterize the rabbit ameloblast phenotype, select differentiation specific markers and utilize them as probes to study enamel gene expression and regulation. First, we will isolate, purify and characterize the rabbit tooth extracellular matrix (ECM) enamel proteins. Our efforts will be concentrated in the 70kDa enamelin complex and the "amelogenin-like" proteins. Two-dimensional gel electrophoresis and HPLC will be used for these purposes. The purified proteins, as well as peptides produced by chemical or enzymatical digestion of the original proteins, will be used for amino acid sequence determination. Once the primary structure of the proteins of interest is known, this information will be used to produce synthetic peptides to define amino acid sequences within the protein molecule. Polyclonal antibodies against the synthetic peptides will be produced. These monospecific antibodies will be used to detect the presence and localization of the specific enamel proteins during tooth morphogenesis. These antibodies will also be of great value to characterize the specific mRNAs and to screen cDNA and genomic libraries produced in expression vectors. In additional studies, we propose to complete the characterization of the mRNAs responsible for the synthesis of these enamel proteins. The total rabbit tooth organ poly(A)-RNA will be used to produce a cDNA library in the expression vector Lambdalgtll. The library will be screened with the monospecific antibodies for enamel proteins and/or with synthetic oligo-nucleotides produced against defined sequences of the enamel proteins. The clones obtained will be further characterized by hybrid arrested and hybrid selected translation. The Lambdalgtll enamel clones will be subcloned in M13 and their nucleotide sequence determined. The enamel cDNA clones will be used as specific probes to determine the structure and organization of the enamel genes by R-loop techniques, nucleotide sequence analysis and Southern hybridization methods. Finally these probes will be used to analyze enamel gene transcription during tooth organogenesis by "in situ" hybridization and immunolocalization at the TEM level.